Most of the modern motorized prostheses are controlled with the surface electromyography (sEMG) recorded on the residual muscles of amputated limbs. However, the residual muscles are usually limited, especially after above-elbow amputations, which would not provide enough sEMG for the control of prostheses with multiple degrees of freedom. Signal fusion is a possible approach to solve the problem of insufficient control commands, where some non-EMG signals are combined with sEMG signals to provide sufficient information for motion intension decoding. In this study, a motion-classification method that combines sEMG and electroencephalography (EEG) signals were proposed and investigated, in order to improve the control performance of upper-limb prostheses.Four transhumeral amputees without any form of neurological disease were recruited in the experiments. Five motion classes including hand-open, hand-close, wrist-pronation, wrist-supination, and no-movement were specified. During the motion performances, sEMG and EEG signals were simultaneously acquired from the skin surface and scalp of the amputees, respectively. The two types of signals were independently preprocessed and then combined as a parallel control input. Four time-domain features were extracted and fed into a classifier trained by the Linear Discriminant Analysis (LDA) algorithm for motion recognition. In addition, channel selections were performed by using the Sequential Forward Selection (SFS) algorithm to optimize the performance of the proposed method.10-channel sEMG + 10-channel EEG, 10-channel sEMG + 20-channel EEG) were obtained with classification accuracies of 84.2 and 87.0%, respectively, which were about 7.2 and 10% higher than the accuracy by using only 32-channel sEMG input.The classification performance achieved by the fusion of sEMG and EEG signals was significantly better than that obtained by single signal source of either sEMG or EEG. An increment of more than 14% in classification accuracy was achieved when using a combination of 32-channel sEMG and 64-channel EEG. Furthermore, based on the SFS algorithm, two optimized electrode arrangements (This study demonstrated the feasibility of fusing sEMG and EEG signals towards improving motion classification accuracy for above-elbow amputees, which might enhance the control performances of multifunctional myoelectric prostheses in clinical application.Institutional Review Board of Shenzhen Institutes of Advanced Technology, and the reference number is SIAT-IRB-150515-H0077.The study was approved by the ethics committee of  Multifunctional prostheses are commonly used by upper-limb amputees to restore their lost motion functions. Surface electromyography (sEMG) is a kind of neural signal that contains motor commands and can be non-invasively extracted on the muscle surface of residual limbs. Due to its relative ease of acquisition and abundant content of neural information, sEMG plays an important role in the control of modern motorized prostheses –4 and reElectroencephalography (EEG) is another kind of neural signal that contains the information related to mental activities of brain but is independent of amputation conditions . SeveralMultiple-source signal fusion is a possible solution for the problem of insufficient information in prosthesis control , 14, wheIn this work, a non-invasive hybrid method was developed, which coherently combined sEMG and EEG signals as a parallel input to classify the upper-limb motions for above-elbow amputees. The proposed method was additionally optimized by channel selections in order to enhance the classification performance with a decreased number of electrodes.Four male transhumeral amputees  were recruited in the experiments, and the demographic information is described in Table Five motion classes of hand open (HO), hand close (HC), wrist pronation (WP), wrist supination (WS), and no movement (NM) were tested, as shown in Fig. REFA 128, TMS International, the Netherlands), where 32 monopolar electrodes were placed on the skin surface of the residual arm for each subject. Based on the residual arm length, electrodes were placed in two different manners: For TH1 and TH2 with relatively shorter residual arms, totally 32 electrodes were distributed in a way shown in Fig. EasyCap, Herrsching, Germany) integrated with a Neuroscan system (Version 4.3). The EEG recording system could automatically mark a vertical line on the EEG recordings as the onset/endpoint of the movement when a target motion picture appeared/disappeared on the screen. And the 64-channel Al-AgCl electrodes were distributed according to the 10–20 system standards as shown in Fig. The sEMG and EEG were simultaneously recorded during the motion performance. The sEMG signals were collected with a high-density sEMG system  [Both the sEMG and EEG acquisition systems used in the experiments have been integrated with a pre-filter by the producers , 18. Thu32-ch sEMG, where only 32 sEMG electrodes were used;64-ch EEG, where only 64 EEG electrodes were used;32-ch EEG, where the first 32 of 64 EEG electrodes were used, locating on the frontal, temporal, and central lobes of cerebral cortex that are associated with thinking and motion functions [unctions , 22, as In this work, the single-signal methods with only either sEMG or EEG, and the dual-signal methods with a combination of both sEMG and EEG, were tested and compared. For the single-signal methods, three channel arrangements were specified:Strategy I (S-I), where 32 sEMG electrodes and 64 EEG electrodes were used, leading to a total of 96 channels of input, i.e. 32-ch sEMG + 64-ch EEG;Strategy II (S-II), where 32 sEMG electrodes and the first 32 of 64 EEG electrodes were used, leading to a total of 64 channels of input, i.e. 32-ch sEMG + 32-ch EEG.For the dual-signal methods, two channel combination strategies were designed:Figure x*, which is determined by the classification accuracy (Acc), into the optimal channel set Sk as the following:q is the total number of channels, k is the number of selected channels. Suppose that k-1 channels have been selected into the optimal channel set Sk-1 by the (k-1)th iteration, in order to select the kth optimal channel x* from the set {X-Sk-1}, each channel within the set {X-Sk-1} was individually combined to the selected optimal channels Xk-1 for motion classification. The channel which achieved the highest classification accuracy was selected as the kth optimal channel, and thereafter added into the Xk-1 for next iteration to obtain a new optimal channel set Xk-1. The iteration procedure was repeated until k increased to the desired level. In this work, 10 optimal sEMG channels were firstly pre-selected from the total 32 sEMG channels by using the SFS algorithm, and then three optimized dual-signal methods combining sEMG and EEG were adopted as follows:Optimized Strategy I (oS-I), where 20 of the 64 EEG electrodes were selected by the SFS algorithm according to Eqs. , where the pre-selected 10 optimal sEMG electrodes were used as the initial channel set S0 in Eq. ,,OptimizeOptimized Strategy III (oS-III), where the pre-selected 10 optimal sEMG electrodes were used as the initial channel set S0 in Eq. ,,OptimizeThe proposed hybrid method combining sEMG and EEG was additionally optimized by decreasing the channel number with the sequential forward selection (SFS) algorithm , 33, 34.32-ch sEMG, 64-ch EEG, and 32-ch EEG, where the average classification accuracies of 77.0, 75.1 and 62.9% were achieved, respectively. Table S-I (32-ch sEMG + 64-ch EEG) and S-II (32-ch sEMG + 32-ch EEG), respectively. By S-I, the classification accuracy for each of the five motion classes was higher than 89%, obtaining an average value of 91.7%, and by S-II, the classification accuracies for different motion classes ranged from 83.9 to 94.1%, resulting in an average of 87.5%. It is clear that the dual-signal methods show much better performances than the single-signal methods both on average and for any specific motion class.Table In order to achieve a satisfactory classification performance with a limited channel number that would be more practical for applications, the dual-signal methods were optimized by decreasing the channel number with the SFS algorithm. Figure oS-I was implemented, where the first 20 EEG channels presented in Fig. oS-I, where the classification accuracy for each motion class was above 81.5% and the average value was 84.6%.Thereafter, the optimized strategy oS-II, the pre-selected 10 optimal sEMG channels marked in Fig. oS-II, the first 20 EEG channels presented in Fig. oS-II, where the mean classification accuracies for all motion classes were above 83% and the overall average value was 87.0 ± 2.7%.To obtain the optimized strategy oS-III, only the first 10 EEG channels presented in Fig. oS-III. For TH1 and TH2 who had left arm amputation, the optimal channels of C6 and CP2 located on the sensorimotor cortex area of the right brain had a higher increase rate in classification accuracy than the channels on the left brain. Moreover, it was found that the EEG channel of POZ which located on the visual cortex area contributed greatly in the classification accuracy increase for both TH1 and TH2. For TH3 and TH4 who had right arm amputation, the optimal EEG channels of C3, CP3, and CZ were located on the motor cortex areas of the left brain, respectively. Tables oS-III, where the mean classification accuracies for different motion classes were all above 78% and the overall average was 84.2 ± 3.0%.To further decrease the channel numbers, in the optimized strategy S-I with 96 channels (32-ch sEMG + 64-ch EEG), the classification accuracy for each subject was over 85% and the average value was 91.7%, which was about 14.7% higher than that for the single-signal method of 32-ch sEMG. By using the optimized dual-signal methods of oS-I, oS-II and oS-III, the total channel number was decreased to 30 or 20; however, the average classification accuracies were still about 7.6, 10.0 and 7.2% higher than those of the single-signal method with 32-ch sEMG, respectively. Especially in oS-III, where only limited channel number of 10-ch sEMG and 10-ch EEG were used, the average classification accuracies of 84.2% was achieved over all the subjects.Figure Combining multiple physiological signals is a possible approach to acquire abundant neural information for prosthesis control, where the motion intentions of above-elbow amputees could be decoded more accurately. To improve the control performances of multifunctional myoelectric prostheses, a hybrid classification scheme using EEG signals as an additional augment to sEMG signals for hand and wrist motion identification is studied in this work.32-ch sEMG input obtains an average classification accuracy of 77.0%, which is 1.9% higher than the 64-ch EEG input and 14.1% higher than the 32-ch EEG input. Thus it might be concluded that the sEMG signals usually outperform the EEG signals in motion classification since the sEMG signals generated by muscle contraction would have a more direct correlation with motions and a higher signal-to-noise ratio in comparison to EEG signals [With the single-signal methods that used sEMG recordings or EEG recordings only for motion classification, the  signals . However signals , 34–36.S-I and S-II) for motion classification. As shown in Table S-I (32-ch sEMG + 64-ch EEG), an average classification accuracy of 91.7% over all the motion classes was achieved. Especially, the classification accuracy for HO was 89.4%, which was 20.5% higher than that by the single-signal method of 32-ch sEMG method. This result has clearly demonstrated that combining some non-EMG signals, such as EEG, to sEMG signals could significantly improve the classification performance for some forearm motions on transhumeral amputees. Different from S-I, the dual-signal method of S-II (32-ch sEMG + 32-ch EEG) used only the first 32 of total 64 EEG channels located on the cerebral cortex area, which is associated with the thinking and motion functions [S-I, by which the classification accuracy was improved by 14.7% (from 77.0 to 91.7%), the S-II could still improve the classification accuracy by 10.5% (from 77.0 to 87.5%), meaning that the first 32 EEG channels contributed the most part of performance improvement.On the other side, the EEG signals produced by mental activities are less dependent on, or even independent of, the amputation conditions. Therefore, EEG signals would be an effective auxiliary signal for sEMG to improve the pattern recognition performances. In this study, sEMG and EEG signals were firstly combined in two different designs , the average classification accuracy of oS-I was 2.4% lower than that of oS-II. A possible reason is that in oS-I, the optimal 10-ch sEMG and 20-ch EEG were selected by using the SFS algorithm from the 32 sEMG and 64 EEG channels, respectively, and therefore they might not be the best matching group for motion classification, which is consistent with the conclusion by Cover [oS-II, the 20 EEG channels were picked out based on the pre-selected 10 optimal sEMG channels, and thus the matching degree between the sEMG and EEG is much higher. In this work, the optimized method oS-II (10-ch sEMG + 20-ch EEG) could obtain an average classification accuracy of 87.0%, which is very comparable to S-II (32-ch sEMG + 32-ch EEG) of 87.5%, but its channel number was less than half of S-II. By oS-III, where totally only 20 channels (10-ch sEMG + 10-ch EEG) were used, the classification accuracy for each subject was above 80.0% with an overall average of 84.2%, which is still about 9.1 and 7.2% higher than those of the single-signal methods of 64-ch EEG and 32-ch sEMG, respectively. These results demonstrated that the proposed strategies have been properly optimized by the SFS algorithm, and the oS-III might be quite desirable for the classification of hand and wrist motions for above-elbow amputees. If higher classification accuracy is desired, the oS-II could be applied. Additionally, the performances of the proposed strategies from TH4 were lower than those for other subjects. A possible reason might be that TH4 has longer and thicker hairs, which leaded to higher impedance between the scalp and the EEG electrodes, resulting in the noisier EEG recordings in comparison to others. So the quality of EEG recordings would be very important for achieving high classification accuracy.There is generally a direct proportion between the motion classification performance and channel number, as demonstrated by the results of this work Figs.  and 6 anby Cover . ContrarFrom Fig. In this study, the results were obtained based on offline analysis, and the performances of the proposed hybrid methods were only evaluated by the classification accuracy. In the future, the efficiencies of hybrid methods, especially the one with optimized channel sets, would be investigated in real-time environment and assessed by more measures such as motion selection time, motion completion time, and motion completion rate , 40. Not10-ch sEMG + 20-ch EEG and 10-ch sEMG + 10-ch EEG were obtained with desirable classification accuracies of 87.0 ± 2.7% and 84.2 ± 3.0%, respectively. The results of this study might be helpful in realizing the control of multifunctional myoelectric prostheses for above-elbow amputees.In this study, hybrid classification methods based on the combination of EEG and sEMG signals were proposed for the classification of hand and wrist motions for above-elbow amputees. By comparing the dual-signal methods with single-signal methods, a significant improvement in classification accuracy was achieved on all the subjects. By using the SFS algorithm, two optimal channel sets of


Doxorubicin (DOX) acts as the model drug, and we researched the capacities of drug loading and release in vitro of the micelles. MTT experiments showed that the blank micelles of PEG-DiHyd-PLA were not cytotoxic to tumor cells  and normal cell (L-02 cells), but the DOX loaded ones displayed more toxicity than the ones without hydrazone, which was consistent to the further confocal laser scanning microscopy and flow cytometry study.In this study, the structure–activity relationship of amphiphilic block copolymer micelles as nanosized drug delivery system was revealed. Firstly, a biodegradable triblock polymers PEG-DiHyd-PLA containing hydrazone bond was synthesized through the ring-opening polymerization. In this method, PEG-DiHyd-Phenol was used as the initiator and L-lactide as the monomer. Then, the polymeric micelles were formed and used as nano-drug carriers with pH sensitivity. The structure and composition of the polymer were characterized by infrared (IR), nuclear magnetic resonance ( In the past two decades, a large number of nanoparticulate drug delivery systems have been extensively explored as in cancer treatment. Some formulations in the form of liposome, polymer–drug conjugates, and micelle particulate have found their applications in clinics and even more are advanced to the stage of clinical trials. Recently, more sophisticated nano-system have been developed to increase the therapeutic efficacy of cancer by controlling of the drug release temporally or spatially. Stimuli-response functionality is becoming increasingly important due to applications in biotechnology and the crafting of smart materials. Stimulus , and poly (anhydride) are core-forming materials and polyethylene glycols (PEG) , N,N′-dicyclohexylcarbodiimide , 4-dimethylaminopyridine , methyl 4-hydroxybenzoate , and hydrazine hydrate aqueous solution  were used as received. Doxorubicin hydrochloride  was stirred with TEA (3 equiv.) in DMSO overnight before the solvent was evaporated using a rotary evaporator to get doxorubicin, i.e., DOX. All organic solvents were analytical reagents and used as received, except that toluene was dried by the sodium method to get anhydrous toluene.Polyethylene glycol  was purchased from Sigma-Aldrich  and dried in a vacuum oven at 70°C before use. L-Lactide , stannous octanoate ; and δ8.09, δ7.92, δ7.84, and δ6.98 (aromatic protons). Meanwhile, PLA-PEG-PLA copolymers without hydrazone linkage were also prepared as pH non-responsive counterpart using PEG as the initiator.PEG-DiHyd-Phenol and L-lactide reacted in anhydrous toluene in the presence of stannous octanoate (100 μL). PEG-DiHyd-PLA with different molecular weights were synthesized by varying the ratio between PEG-DiHyd-Phenol and L-lactide. The reactions were conducted at 110°C under the protection of nitrogen gas for 24 h. The copolymer was precipitated three times with cold ethyl ether. It was dried at 45°C in vacuum. PEG-DiHyd-PLA were obtained with a yield of 72%. An AVATAR360  and an AVANCE 400 spectrometer  were used to determine the chemical structure of the polymers. A Damn Eos  gel permeation chromatograph (GPC) instrument equipped with Phenogel 10E6A column and an OPTILAB rEX refractive-index detector was used to determine the molecular weight and polydispersity. Tetrahydrofuran (THF) was used as the eluent at a flow rate of 1.0 mL/min at 30°C and polystyrene standards for the calibration. A Zetasizer Nano-ZS90  and Transmission electron microscopy (JEM-100CX II TEM) were employed to determine the size and the morphology of the micelles.PLA-PEG-PLA or PEG-DiHyd-PLA (30 mg) dissolved in THF 2 mL was added into 40 mL pure water by dropwise. The mixture was stirred for 36 h at room temperature. The sizes evolution in different solutions of the micelles were measured to determine the pH sensitivity. Ten milliliters freshly prepared micelle dispersions was adjusted to pH 5.0 or pH 4.0. The sizes were measured on DLS after 24 h incubation at 37°C with shaking. CMC was determined using pyrene as a fluorescence probe  and DOX (2 mg) were dissolved in 1 mL acetone. The solution was added dropwise into 30 mL pure water stirring. Then, the micellar solution was dialyzed against water for 36 h. After filtered through a 0.22 μm syringe filter to remove undissolved DOX, DOX-loaded micelle dispersion was freeze-dried. The drug loading content (DLC was determined by the measurement of fluorescence of DOX (excitation wavelength at 481 nm and emission wavelength at 558 nm). DLC was calculated by the formula below:in vitro release experiments of DOX were conducted at 37°C. Dialysis bag  filled with 3 mL micellar solution was sealed and immersed in 40 mL buffers solution. Three buffer solutions were used: acetate buffer , acetate buffer , and PBS . At desired time intervals, 4 mL of solution outside was taken out for fluorescence measurement. Meanwhile, 4 mL fresh medium was replenished. Cumulative released DOX was calculated according to following formula:The Er means cumulative release of DOX (%); Ve means volume to be taken very time (mL); V0, the volume of medium (mL); Ci, concentration when certain volume to be taken (μg/mL); mdrug, total mass of DOX contained in the release system (μg); n, sampling times.In this equation, 2) on a 96-well plate. The cell density is 5 × 103 cells per well in 100 μL of 1640 medium (containing 10% FBS). 24 h later, the medium in each well was removed and 100 μL DOX-loaded micelles or free DOX solution were added into the wells. Each concentration has four replicates. Each sample was performed in quintuplicate. After incubation for 48 h, and the viability of cells was measured using the methylthiazoletetrazolium method. Cell viability (%) was calculated by the following equation . Cells were seeded and incubated for 24 h .where A4 cell using RPMI-1640 medium supplemented with 10% FBS. 24 h later, free DOX, DOX-loaded PEG-DiHyd-PLA, and PLA-PEG-PLA micelles were added into the wells at the same DOX concentration of 10 μg/mL. After being incubated at 37°C for 3 and 12 h, the cells were washed with PBS and fixed with PBS containing 4% formaldehyde for 15 min at room temperature. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min. The fluorescence signals of DOX and DAPI staining were investigated and imaged by the confocal laser scanning microscopy system (CLSM).HepG-2 cells were seeded on the cover slips in culture dish with a density of 8 × 105 cells) in 1640 media and incubated for 24 h at 37°C. Then, HepG-2 Cells were treated with 1.5 mL of fresh cell culture medium and containing free DOX and DOX-loaded nanoparticles  were added. After 48 h of incubation, the drug containing media was collected, and cells were trypsinized (without EDTA), centrifuged, washed with Binding Buffer (1X) for two times. Then collect the cells, and stained with Annexin V-FITC and propidium iodide (PI) for 20 min following the operating instructions (In the dark environment). Finally, the sample was tested by flow cytometry within 1 h.DOX-loaded micelles and free DOX uptake HepG-2 cells was assessed using the flow cytometry cell analyzer. HepG-2 cells were seeded in culture dish , and δ4.52 (–COOCCHO–). Through the above analysis, we can confirm the success of the synthesis of CHO-PEG-CHO. 1H-NMR showed that the macroinitiator PEG-DiHyd-Phenol was successfully synthesized, as indicated by the signal at δ8.41, which was assigned to hydrazone protons (Ar-CH = N)  Figure . In the ) Figure , charact1H-NMR spectra. The calculated values based on 1H-NMR spectra were consistent with the designed ones. Gel permeation chromatography (GPC) measurements confirmed a unimodal distribution of Mn  and narrow distribution . Therefore, well-defined biodegradable triblock polymers PEG-DiHyd-PLA was successfully synthesized.Molecular weights of synthesized polymers are listed in Table in vivo, which is so important for the micellar drug delivery system.Micelles were formed by adding the amphiphilic polymers PEG-DiHyd-PLA into aqueous solution. The micelles have diameters ranged from 70 to 130 nm and increase with their molecular weight. The average particle size of the drug loaded micelles was larger than that of the blank micelles, which may be due to the larger volume of micelles after the hydrophobic core of the drug loaded with the micelles . DOX was successfully loaded into the micelles and presented a more rapid and complete drug release in acidic condition (pH 5.0). The results of PQ, performed the synthesis and characterizations; XW, performed the cell experiments; LL, designed the synthesis and wrote the paper; HY, performed the formulations; SS, designed the whole experiment.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Major depressive disorder (MDD) is a common condition that afflicts the general population across a broad spectrum of ages and social backgrounds. The inflammatory hypothesis of depression posits that immune hyperactivation and dysregulated cytokine production are involved in depression. To investigate cytokine profiles in patients with MDD, we examined the levels of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α, and those of the anti-inflammatory cytokines IL-10 and transforming growth factor (TGF)-β1 in antidepressant drug-naïve patients with MDD. Compared to healthy controls, patients with MDD had significantly higher levels of IL-1β, IL-10, and TNF-α, but significantly lower levels of IL-8. There were no significant differences in the levels of IL-6 or TGF-β1. We found linear correlations between IL-1β, TNF-α, and IL-8, and the severity of depression, as well as between IL-8 and anxiety level in patients with comorbid anxiety disorder. In addition, higher IL-1β and TNF-α levels were associated with higher Hamilton Depression Rating Scale (HAMD) scores, while higher IL-8 levels were associated with lower HAMD and Hamilton Anxiety Rating Scale scores. Here we present evidence of changes in cytokine levels in antidepressant drug-naïve patients with MDD. Abnormal expression of inflammatory cytokines in patients with depression suggests that depression activates an inflammatory process. Immunological abnormalities may be involved in the pathophysiology of depression. Major depressive disorder (MDD) is a common condition that afflicts the general population across a broad spectrum of ages and social backgrounds, and regardless of sex. MDD is a devastating mental illness, and is characterized by a lifetime prevalence of >16% . This main vivo and in vitro studies using the same agent have had different results [Antidepressants have a variety of effects on cytokine levels in MDD , 9. Howe results , 11. TheMDD is triggered by stress in a major subgroup of patients. Chronic stress, such as early life stress or separation stress, has been implicated in dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis , which bDiagnostic and Statistical Manual of Mental Disorders, 5th Edition[Patients with MDD were selected from adult outpatients admitted to the Department of Psychiatry at Tongji Hospital  between December 2015 and January 2017. Diagnosis of MDD was based on the patients meeting a number of qualitative diagnostic criteria outlined in the h Edition  were collected at 08:00 AM by venipuncture. The samples were left standing for 30 min and then centrifuged  for 8 min. A total of 1 mL of the supernatant (serum) was collected for quantitative assays. Serum levels of IL-1β, IL-6, IL-8, IL-10, TNF-α, and TGF-β1 were analyzed using standard capture enzyme-linked immunosorbent assay kits (Xinbosheng Biotechnology). Cytokine analysis was performed using an enzyme marker analyzer . Detection was performed using a bead-based antibody-antigen sandwich technique. Briefly, specific binding occurred between cytokines and monoclonal mouse anti-cytokine antibodies pre-coated on the enzyme-linked immunosorbent assay plates. Expression levels were determined following incubation with polyclonal anti-cytokine antibodies conjugated to alkaline phosphatase  in 7.5 mL buffer solution. Binding of the secondary antibodies enabled the production of detectable photoelectrons by the luminescent substrate. The results are presented as concentrations of cytokines (pg/mL) in serum. To ensure the accuracy and repeatability of the experiments, all samples were tested in the same run, which included a set of standards measured in duplicate. Our calculations indicated that the coefficients of variability in all groups were less than 10%. The intra-assay and inter-assay coefficients of variability were also lower than 10%.All statistical analyses in this study were performed using SPSS version 17.0  based on a significance level of α = 0.05. For quantitative variables , mean concentration was used to describe the centralized trend and standard deviation was used to describe the discrete trend. One-way analyses of variance were used to examine quantitative differences between patients and healthy controls. The chi-squared test was used to examine differences in qualitative variables  between the two groups. Simple linear correlation analysis was used to estimate correlations between quantitative variables and HAMD or HAMA scores. Multiple linear regression analysis was used to analyze the influences of multiple factors.p < 0.01) and significantly lower levels of IL-8 (p < 0.01). There were no significant differences in the levels of IL-6 or TGF-β1 (p < 0.05) (p < 0.05). Higher IL-1β and TNF-α levels were associated with higher HAMD scores, while higher IL-8 levels were associated with lower HAMD scores (p < 0.05) such that higher IL-8 levels were associated with lower HAMA scores  . MultiplD scores . MultiplA scores .Previous research has suggested that inflammation may be an etiological link between psychological stress and MDD , 22. In Changes in the pro-inflammatory cytokine profiles were inconsistent in our antidepressant drug-naïve patients with MDD. One plausible interpretation of this finding is that the autonomic nervous system may not be implicated as a significant underlying mechanism in MDD. It has been reported that IL-1β can lead to HPA axis over-activation . TNF-α cWe found that the levels of anti-inflammatory cytokines, such as IL-10, increased in the patients with depression . SimilarWe lower levels of IL-8 in the patients with MDD, and the IL-8 level was associated with the degrees of anxiety and depression in the patients with depression . IL-8 isThe changes in IL-6 levels were inconsistent in the patients with MDD ,39, but Similar to IL-6 levels, the expression levels of TGF-β1 in patients with MDD were inconsistent , 44. We Our study has several limitations, the first of which is the relatively small sample size and the absence of an analysis of the depressive disorder type. Second, although we studied various cytokines in this investigation, our results do not reflect changes in the entire immune network response, such as anomalies in immune cells .Future studies should include analyses of TH1, TH3, and other immune cells, which may provide a comprehensive understanding of the changes in the immune network response. Third, there is evidence suggesting that peripheral cytokine levels are associated with cytokine levels in the brain. Increased cytokine levels in the blood of patients who have not undergone surgery are accompanied by increased cytokine levels in cerebrospinal fluid. The expression of central cytokines may also be induced by peripheral cytokines, suggesting that cytokine levels in the blood reflect changes in cytokine levels in the brain, at least to some extent ,47. HoweOur findings demonstrate that antidepressant drug-naïve patients with MDD exhibit abnormal immune regulation and immune system activation. We discussed the cytokine hypothesis, which posits that depression is a process of inflammation enhancement. However, not all serum cytokines had increased levels or (necessarily) participated in immune activation in the patients with depression. Nevertheless, immune activation was related to disease severity. Specific mechanisms may be involved in the complex network structure of cytokines.


Recently, a modified insulin-like growth factor-1 (IGF)–Child-Turcotte-Pugh (CTP) classification was proposed to improve the original CTP classification. This study aimed to validate the new IGF-CTP classification system as a prognostic maker for patients with hepatocellular carcinoma (HCC) in a hepatitis B virus endemic area.We conducted a post-hoc analysis of a prospective cohort study. We used Harrell’s C-index and U-statistics to compare the prognostic performance of both IGF-CTP and CTP classifications for overall survival. We evaluated the relationship between HCC stage and the four components of the IGF-CTP classification  using nonparametric trend analysis.P = .07 by U-statistics). A lower serum level of IGF-1 was related to a more advanced cancer stage (P < .01). The remaining components of the IGF-CTP classification were not significantly related to tumor stage .We included a total of 393 patients in this study. In all, 55 patients died during the median follow-up of 59.1 months. There was a difference between IGF-CTP class and CTP class in 14% of patients. Overall, the IGF-CTP classification system had a higher prognostic value  than the CTP system , but the difference was not statistically significant (The IGF-CTP classification was slightly better than the original CTP classification for predicting survival of patients with HCC in a chronic hepatitis B endemic area. This is most likely due to the fact that serum IGF-1 levels reflect underlying HCC status. Most cases of hepatocellular carcinoma (HCC) occur in the setting of chronic hepatitis and liver cirrhosis. Because of these co-existing underlying liver diseases, the functional reserve of the liver, in addition to tumor status, is a major prognostic factor in patients with HCC. The Child-Turcotte-Pugh (CTP) classification system has been the most widely accepted prognostic model for evaluating functional hepatic reserve. The CTP et al constructed a modified CTP classification system (IGF-CTP) to predict survival in patients with HCC by replacing the two subjective parameters in the traditional CTP system with IGF-1 level (Insulin-like growth factor (IGF)-1 is a hormone that is mainly synthesized by the liver. IGF-1 plays a critical role in childhood growth and anabolic metabolism. Baseline-1 level .10] The Theet alThe aims of the current study were 1) to validate the IGF-CTP classification system in patients in a CHB endemic area by comparing the accuracy of the IGF-CTP and the CTP classifications for assessing overall survival (OS) after the first treatment for HCC and 2) to clarify the causes of differences between the predictive values of the classification systems.We included patients who were enrolled in an ongoing prospective cohort study to identify biomarkers for predicting the prognosis of HCC at Seoul National University Hospital . Briefly, between January 2006 and December 2012, 409 patients who were newly diagnosed with HCC were recruited to the prospective cohort. HCC was diagnosed on the basis of recommendations of the American Association for the Study of Liver Diseases.[Peripheral venous blood samples (5 mL) were collected from all patients before initial treatment for HCC. All sera were aliquoted and snap-frozen at -80°C until analysis. Serum IGF-1 levels were measured using an immunoradiometric assay according to the manufacturer’s instructions .The IGF-CTP score comprises four laboratory values: total bilirubin, albumin, PT, and IGF-1. Cut-off The primary endpoint of this study was to compare the prognostic performance of both classification systems for OS. OS was defined as the time from the date of diagnosis of HCC to the date of death from any cause. The secondary endpoints included OS prediction according to combination of both CTP and IGF-CTP classifications and the association of each parameter in the IGF-CTP classification with tumor stage. The cut-off date for data collection was September 30, 2015, and there were no missing values for these analyses.P-value of 0.05 or less was considered statistically significant. Pearson’s χ2 test was used to analyze differences among groups of categorical data. Survival curves were estimated using the log-rank test. Associations between insulin-like growth factor 1 and OS, and between both scoring systems and OS were assessed using univariate and multivariate Cox proportional hazard models. Harrell’s C-index was calculated to compare the prognostic power of each classification system in our cohort. A nonparametric trend analysis was used to evaluate possible trends in the scores of each component in the IGF-CTP classification  related to underlying tumor status.[P-values used in the nonparametric trend analysis are based on z-statistics.Statistical analyses were conducted with SPSS 22.0 , the R statistical programming environment, Version 3.2.0 , and Stata 13.1 . A two-sided We included a total of 393 patients who were newly diagnosed with HCC in the final analysis. The mean age of the patients at the time of diagnosis was 56.8 ± 9.5 years and most of the patients (77.9%) were male. CHB was the most common cause of underlying liver disease (78.9%). According to the CTP score, 334 (85.0%), 57 (14.5%), and 2 (0.5%) patients were classified as having class A, B, and C disease, respectively .P < .01) and high  IGF-1 levels. Patients with intermediate IGF-1 levels also had a worse outcome than those with high IGF-1 levels. However, the difference was not significant  , 55 (14.0%) patients died; the median OS was not reached. The 5-year survival rate was 84.1%. Patients with low IGF-1 levels (< 26 ng/mL) had a significantly worse prognosis than patients with intermediate  .P = .07) (P = .21).OS was significantly associated with different hepatic reserve classes according to both classification systems . The obsP = .07) . The BarWe analyzed the differences in patient distribution of the two classification systems . For 86.P = .02 for AB group; HR = 82.25, 95% CI = 10.22–710.85, P < .01 for AC group). A total of 306 (91.6%) patients were in the AA group; this group had a 5-year survival rate of 87.0%. The AB group included 27 (8.1%) patients; this group had a lower 5-year survival rate (65.5%) than the AA group. CTP class B patients who were reclassified as IGF-CTP class C (BC group) also had a worse prognosis than patients classified as class B by both the CTP and the IGF-CTP classification systems (BB group) .According to our results and the Kaplan-Meier curves , the IGFP < .01). Total bilirubin (P = .11), albumin (P = .33), and PT (P = .39) were not related to AJCC stage.Although the predictive value of the IGF-CTP classification system was not significantly better than the original CTP system, the C-index of the IGF-CTP classification was higher than that of the CTP classification in our study. To clarify the mechanism of improvement of the predictive value, the associations between AJCC stage and each parameter included in the IGF-CTP classification were analyzed . AccordiWe validated the predictive value of the IGF-CTP classification system for assessing survival in HCC patients from a CHB endemic area. The C-index of the IGF-CTP classification was higher than that of the original CTP classification, but this difference was not significant. The proportion of patients who had a difference in risk stratification between the two classifications was not enough (14.0%) to establish statistical significance. Lower serum IGF-1 levels were significantly associated with more advanced HCC. These findings suggest that the IGF-CTP classification system has a tendency to predict survival more accurately than the CTP system by reflecting underlying HCC status.Functional hepatic reserve can affect survival of HCC patients, so several HCC staging systems include CTP class when assessing disease stage.–17 HowevInterestingly, recent studies suggested that higher plasma IGF-1 levels were related to various malignancies, including prostate, breast, colon, and lung cancers.–29 HowevThis was the first study to evaluate the long-term predictive value of the IGF-CTP classification system for survival in HCC patients. A major strength of our study is that this is the first validation of the IGF-CTP classification system in a CHB endemic area. In our cohort, 78.9% of total patients were infected with HBV; most patients included in previous validation cohorts were infected with HCV., 11 Furt10,11 Since most patients (86.0%) were designated as the same risk class by both classifications, it was impossible to establish the statistical significance of the differences between the two classification systems. The negative results of our study might originate from the fact that many of the enrolled patients had minimal risk factors for a poor HCC prognosis, rather than differences in the etiology of HCC. However, the limited discriminative function of the IGF-CTP classification in patients with mild disease is a critical weak point, considering recent trends in HCC surveillance.Our study has some limitations that should be considered. First, the majority of patients (77.9%) had well-compensated hepatic reserve and were classified as having class A disease by both the CTP and the IGF-CTP classifications. The proportion of patients with this “AA” designation was much higher in our study than in previous validation studies.Although its improvement was not statistically significant compared to the original CTP classification, the IGF-CTP classification system demonstrated better discriminatory function for predicting survival of patients with HCC in a CHB endemic area. The IGF-CTP classification also considerably improved the accuracy of survival prediction in previous validation studies. Therefore, using the IGF-CTP classification instead of the CTP classification in clinical practice can help physicians properly classify patients regardless of the etiology of HCC.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Based on the images generated from two multi-slice computed tomography (CT) scanners, we intended to compare the five-line sign of normal interlobular fissures produced on axial or oblique maximum intensity projection (MIP) reconstructions using different algorithms.Two groups of 50 subjects underwent either 16-slice or 256-slice spiral unenhanced chest CT. None of them in either group displayed any abnormality. For each case, maximum intensity projection (MIP) data were used to calculate the axial or oblique projection using four algorithms: standard axial, standard oblique, high-resolution axial, and high-resolution oblique algorithm. The results were then used to reconstruct images of six locations of the lung. The clarity of the five-line sign of the reconstructed MIPs for the interlobular fissures was determined and graded as 1 (unclear), 2 (barely clear), or 3 (clear). Comparisons of the rate and the degree of clarity were performed using non-parametric tests.p < 0.001), whereas the high-resolution axial algorithm was the worst. In addition, the two CT units exhibited no significant differences in the clarity of the five-line sign (p = 0.273).Data from both the 16-slice and 256-slice CT revealed that the standard oblique algorithm was the best among the four methods for presenting clear images of the five-line sign (The standard oblique algorithm was the best approach to reveal the five-line sign of normal lung fissures. Both 16-slice and 256-slice CT were effective for reconstructing the sign. Both 16-slice and 256-slice CT were effective for reconstructing the five-line sign of normal interlobular fissuresThe standard oblique algorithm was the best approach to reveal the five-line signThe MIP-rendered five-line sign has smooth lines and clear gaps and can, therefore, be used as the referencing object for other planar structures in the lungMulti-slice spiral computed tomography (CT)-based thin reconstructed images facilitate the clear presentation of various structures and lesions in the lung from any angle –3. ReconUsing a 64-slice spiral CT scanner, we previously noticed that normal interlobular fissures presented as multiple parallel shadow lines in MIP-reconstructed images, while the number of shadow lines was equal to the ratio of the thickness of the MIP reconstruction layer to the thickness of the original layer. A five-line sign could be generated if the following parameters were adopted: 1.25-mm layer thickness, 1.25-mm space, and 6.25-mm MIP slab thickness [In this study, we examined the effect of two different CT units (16-slice or 256-slice), using 1.0-mm reconstruction layer thicknesses, 1.0-mm space, and also added a high-resolution reconstruction algorithm on the clarity rate and level of the five-line sign.This retrospective study was approved by the institutional review board  with a waiver of informed consent. Candidates were selected based on a consecutive series of apparently normal unenhanced CT examinations performed from December 2012 to April 2013. We included 128 subjects who had no abnormalities on a spiral chest CT scan. Among these, 62 were scanned using 128-row 256-slice CT; 12 of these subjects who had poor-quality images (n = 9) or underdeveloped interlobular fissures (n = 3) were excluded from the study. The remaining 50 subjects included 35 men and 15 women aged from 18 to 77 years. Sixty-six individuals were scanned using a 16-row 16-slice CT unit; 16 of these subjects who had poor-quality images (n = 12) or underdeveloped interlobular fissures (n = 4) were excluded from the study. The remaining 50 subjects included 28 men and 22 women aged from 18 to 75 years. Here, underdeveloped interlobular fissures means that any segment of the oblique fissures or the major, such as upper, middle or lower one was absent; poor image quality means that there was movement artefact of the bronchi, vessels, fissures and diaphragm on coronal reconstruction in the lungs.The 256-slice CT unit was a Philips  Brilliance iCT . Technical parameters included a collimation width of 128 × 0.625 mm, a rotation speed of 0.75 s, and a pitch of 0.993. Image reconstruction was performed using two kernels: the standard kernel for the standard algorithm and the lung-enhanced kernel for the high-resolution algorithm. The 16-slice unit was a Siemens  Somatom Sensation 16 . Technical parameters included a collimation width of 16 × 0.75 mm, a rotation speed of 0.5 s, and a pitch of 1.1. The scanner was equipped with two kernels: the B40f kernel for the standard algorithm and the B80f ultra-sharp kernel for the high-resolution algorithm. Both CT units were operated under the following common parameters: voltage 120 kVp; tube current 200 mAs (with tube current modulation active on the 16-slice CT unit); 1.0-mm reconstruction slice thickness; 1.0-mm space; 5.0-mm axial MIP thickness; 6.0-mm oblique MIP thickness; and lung visualisation window (width 1000 Hounsfield units (HU), level − 700 HU).For multi-planar MIP reconstruction, the 1-mm thin-section-based reconstruction data from the 100 patients were imported into a Brilliance™ Workspace Portal  to perform multiplanar and MIP reconstruction.Two orientations were adopted for MIP reconstruction: axial and oblique . Specifically, reconstruction tangent lines were rotated clockwise or counter clockwise on the sagittal plane until the five-line sign of the pleural segment was clearly displayed on the MIP-reconstructed coronal oblique plane. The angle between the fissure and the tangent line was positive as the tangent line was rotated clockwise from 0°; otherwise, the included angle was negative as the tangent line rotated counter clockwise from 0°; “+” and “-” was marked apart and each of them represented a given direction of the tangent line rotated initially, but did not indicate how small or large the degree of the angle was Figs.  and 2. BEach sample was subject to calculation using the standard axial algorithm, the standard oblique algorithm, the high-resolution axial algorithm, and the high-resolution oblique algorithm to reconstruct images at six locations in the lung: upper left, upper right, middle left, middle right, lower left, and lower right. Two physicians, Y.Z. and A.Y., with 5 years and 20 years of diagnostic imaging experience, respectively, worked in consensus to formulate an image clarity standard for the five-line sign of interlobular fissures in the MIP-reconstructed images.Based on this standard, the axial and oblique rendering outcomes of the 100 subjects who had normal interlobular fissures were assessed. The clarity levels of the five-line sign were graded as 1 , 2 , or 3   clarity levels for the four calculation methods and the two CT units as well as the clarity rates of the five-line sign among six locations on axial or oblique positions. The significance level of the statistical analyses was set as α = 0.050. Inter-observer agreement for clarity levels of the five-line sign from different CT scanners and different algorithms was estimated by using weighted κ statistics [Data analysis was performed using SPSS 16.0 software . The Kruskal-Wallis test was used to compare the clarity levels of the five-line sign and the angles between the fissures and tangent lines derived from the two CT units and the four calculation methods. The Kruskal-Wallis test and a subsequent Mann-Whitney atistics .The following settings were used for both CT units: 1.0-mm reconstruction thickness, 1.0-mm space and 5.0-mm MIP slab thickness. Correspondingly, both units generated MIP reconstruction images at the axial position that revealed parallel five-line shadow lines for the normal pleura. In other words, the number of shadow lines was equal to the ratio of the thickness of the MIP-layer to the thickness of the original layer Fig. . Howeverp < 0.001), whereas the high-resolution axial algorithm presented the lowest clarity (p < 0.001) among the four methods. There were no significant differences in the clarity of the five-line sign using the two CT devices when the same calculation method was used . In the lower left zone, however, 256-slice CT yielded significantly superior results to 16-slice CT . If data from level 2 (barely clear) were included, the total clarity rates of the six locations increased to 100% and 97.3% for 16-slice CT and 256-slice CT, respectively.When the standard oblique algorithm was used, the rates of assignment to clarity level 3 from six locations were 70.3% for the 16-slice CT and 74.3% for the 256-slice CT, which were not significantly different  in the lower right lung area. In addition, there were no significant differences between the two CT scanners in the clarity of the five-line sign at any location when calculated using the same methods. The clarity rates of the five-line sign derived from the different methods and the two CT devices are summarised in Table When the high-resolution oblique algorithm was used, 256-slice CT exhibited a higher rate of assignment to clarity level 3 than did 16-slice CT .In conclusion, the five-line sign of interlobular fissures can be revealed in MIP reconstructed images derived from different CT scanners. The standard oblique algorithm generated the best reconstruction outcome, whereas the clarity levels were not significantly different between the two CT scanners. The MIP-rendered five-line sign of normal interlobular fissures can be used as the referencing object for other planar structures in the lung.


Neurotrophin-3 (NT-3) is a neurotrophic factor that mainly binds to the tyrosine kinase C (trkC) receptor. NT-3 has been shown to have neuroprotective effects in focal cerebral ischemia. Exercise also has ability to induce functional recovery in focal cerebral ischemia. However, the relationship between NT-3, its receptor trkC, and exercise has not been revealed. In this study, we assessed the expressions of NT-3 and trkC in focal cerebral ischemia. We also assessed the expression of NT-3 and trkC with treadmill exercise in focal cerebral ischemia. The results showed that, in a permanent middle cerebral artery occlusion rat model, exercise increased NT-3 and trkC expression. However, the patterns of expression of NT-3 and trkC at different time points varied. These results suggest that exercise-induced functional recovery in focal cerebral ischemia was related to NT-3 and trkC, but the role on times of NT-3 and trkC differed, although trkC is the receptor kinase for NT-3.  Stroke is the fifth leading cause of death in the United States, with approximately 800,000 people a year having a stroke, or one every 40 seconds . Stroke Despite numerous trials to ameliorate stroke, there have been no satisfactory outcomes. However, exercise therapy after stroke has long been considered a logical candidate to ameliorate physical disability . AlthougMany studies have also demonstrated that neurotrophic factors play important roles in neuronal survival, proliferation, maturation, and outgrowth in the developing brain and neuroprotective function in mature brain insult . These sNeurotrophin-3 (NT-3) is one of the neurotrophic factors, which comprise a family of proteins that includes nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), NT-3, and neurotrophin-4 (NT-4). The neurotrophic factors function via interactions with different members of tyrosine kinase (trk) receptors . Each neIn this study, we investigated the effects of treadmill exercise on the expression of NT-3 and trkC in a rat model of experimental cerebral infarction.n = 4 for each of the three groups) Protocols including procedures for use of animals and the care were approved by Catholic University Animal Care Committee according to the compliance guidelines.In this study, we used 59 adult Sprague-Dawley rats. All of them were male with body weight ranging from 275 to 325 g for each rat. Female rats were not included in this study to avoid bias due to sexual difference. Rats were divided into several groups, such as middle cerebral artery occlusion (MCAO) group as well as sham-operation as a control. There were 35 rats in MCAO group and 12 rats as control in number. The MCAO group was further divided into two subgroups with or without exercise in two days. 18 of them had exercise and 17 of them did not have exercise; thus we define these subgroups as “exercise versus nonexercise.” Garcia scale was used to determine the degree of severities. Garcia scale contains six items (spontaneous activity/symmetry of spontaneous movements/symmetry of forelimbs during movement/climbing the wire cage/reaction to touch/response to vibrissae touch). The higher score were the better performance. We measured total score to further define the range of severities. The total score ranged from 3 to 18. We subcategorized into mild group , moderate group , or severe group as III with the score of less than 7 (including 7) –8. In ad2 and 70% N2O was used for induction. Isoflurane (1.5%) was used for maintenance. Following anesthesia, the left common carotid artery was exposed through a midline cervical incision. As next step, external carotid artery (showing level at bifurcation as branches) was coagulated. And then, ligation of the pterygopalatine artery was performed with a 5.0 silk suture. Nylon monofilaments (4.0) were used for the occlusion. For occlusion procedure, the monofilament tip was rounded by heating and was inserted into the level of bifurcation site of carotid artery. By advancing 16–18 mm, it can occlude the origin and proximal part of the anterior cerebral artery. We closed the wound with monofilament being in place. Following these procedures, rats were allowed freely to food and water. Rectal temperature was maintained at 37 ± 1°C. We used a thermistor-controlled heating blanket [For the experimental focal cerebral infarction model, this method had been used according to what was previously described elsewhere . InitialTo further access the effect of exercise, we applied the treadmill . This treadmill exercise was started in two days following the operation, not immediately. In exercise program, on the first exercise day, 10 m/min was given. We increased gradually up to 15 m/min on the second day. On the third and subsequent days, exercise set was 20 m/min. The tilting angle of the exercise table was maintained and set to 0°. This protocol was performed every 30 minutes for 12 days –8.μm using a sliding microtome. For blocking, 10% normal goat serum (NGS), 1% bovine serum albumin (BSA), 0.2% Triton X-100, and 1% H2O2 were used in PBS. Following washing with PBS (×3), anti-NT-3  and anti-trkC  antibodies were used in 10% NGS and 1% BSA for overnight at cold room with temperature of 4°C. We used DAB kit  for immunoperoxidase labeling. They were evaluated and captured using an BX51 microscope  [At day 16, rats were anesthetized first and then sacrificed. For transcardiac perfusion, heparinized saline and then 4% paraformaldehyde in phosphate-buffered saline (PBS) were perfused. Sections were cut at a thickness of 30 , Japan) .μg, was loaded and separated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used with 10% polyacrylamide and 0.05% bis-acrylamide [β tubulin  was checked on the sample membrane. We detected signals with enhanced chemiluminescence , using autoradiogram by exposing 10 to 30 min [Following anesthesia, the brains were removed from the skull and these were dissected into the right or left hemispheres. For protein extraction, they were placed in 10 volumes of cold homogenization buffer  with protease inhibitors  being freshly added. Tissue then was homogenized by sonicator. Concentrations of protein were checked by the Bradford method . By adding the sampling buffer, equal amount of protein, 20 rylamide . By sepao 30 min –8.p < 0.05 were considered to be statistically significant. We triplicated experiments to see whether the findings were replicated or not.The Mann–Whitney test and Kruskal-Wallis test were used for groups comparison. We used SPSS (ver. 12.0). NT-3 exists in either a dimer (40 kDa) or a monomer form (21 kDa) protein. Exercise increased the immunoreactivities of monomer protein in both hemispheres of the sham-exercise group. In the ischemia group, exercise led to increased signals in the contralateral hemisphere .p = 0.027). In the ipsilateral region, expression of the NT-3 dimer decreased at the time point of postinfarct day 9, while enhanced immunoreactivities were observed on postinfarct day 23 (p = 0.027). In the ischemia-exercise group, expression of the NT-3 dimer was decreased at postinfarct day 16 but increased again at postinfarct day 23   .p = 0.027, ipsi: p = 0.050). However, in the ischemia-exercise group, expression of NT-3 dimer protein decreased these signals  (The expression of the NT-3 dimer increased in severities II and III in the ischemia-nonexercise group (contra: = 0.027) .Immunohistochemistry showed that immunoreactivity decreased in the infracted area but increased in and around the infarct core region in the ischemia-nonexercise group. However, exercise appeared to restrict immunoreactivity only in and around the ischemic region .trkC exists either as a full-length (140 kDa) protein or as a truncated (90–95 kDa) protein. Exercise increased the two forms of trkC in the sham group. In the ischemia-nonexercise group, both forms of trkC were increased in the ipsilateral region. In the ischemia-exercise group, these immunoreactivities were decreased in the ischemic ipsilateral region, while they were increased in the contralateral nonischemic region .p = 0.027, truncated: p = 0.027). In the ischemia-exercise group, expression of the full-length form increased with time in the contralateral region (p = 0.027). Conversely, expression of the full-length form decreased in the ipsilateral region. The truncated form was increased at 23 days after infarct on the contralateral nonischemic region (Time-dependent patterns showed that both forms of trkC were increased at postinfarct day 23 in the ischemia-nonexercise group. In the ipsilateral region, both forms of trkC increased with time (full: c region .Immunohistochemistry showed that exercise increased immunoreactivity in both hemispheres of the sham group, particularly in the vascular structures, and that exercise also concentrated immunoreactivity around the ischemic region in the ipsilateral hemisphere .In previous studies, we confirmed the expression of BDNF/trkB, NGF/trkA, and NT-4/trkB following focal cerebral ischemia with exercise. Interestingly, exercise increased the level of BDNF/trkB, NGF/trkA, and NT-4/trkB in the contralateral hemisphere; however the time points were different according to the neurotrophic factors and their receptors –8. BasedMany studies have been conducted to characterize neurotrophin –14. AmonPrevious studies showed that exercise has neuroprotective effect and can increase neurogenesis. The duration and intensity of exercise are important factors promoting the plasticity and enhancement of the brain. Indeed, progressive exercise like walking on a treadmill was sufficient to improve brain function , 15, 16.Although the mechanism of neuroprotective effect of NT-3 is not completely understood, several studies have shown that NT-3 binds to high affinity receptor trkC, triggers the receptor, and signals cascades. These intracellular signaling pathways modulate gene expression and are responsible for most of the neuroprotective effects related to neuronal growth, survival, and differentiation –19.In this study, we showed that the expression of trkC was also increased by treadmill exercise itself. However, in focal cerebral ischemia, exercise decreased the expression of trkC in ipsilateral region. Moreover, the expression of trkC gradually increased by times without exercise in focal cerebral ischemia. These results are contrary to the expression of NT-3. Exercise increased the expression of trkC, as for NT-3, in the contralateral region, but not in the ipsilateral region in focal cerebral ischemia.Overall, the results of this study indicate that exercise induced functional recovery in focal cerebral ischemia and that this effect was related to NT-3 and trkC. However, the roles of NT-3 and trkC likely differed based on the observed differences in the timing of their expression. Therefore, although trkC is the receptor kinase for NT-3, expression profiles of NT-3 and trkC are not directly matched among the time points of exercise-induced functional recovery.


Cognitive reserve, the brain’s capacity to draw on enriching experiences during youth, is believed to protect against memory loss associated with a decline in hippocampal function, as seen in normal aging and neurodegenerative disease. Adult neurogenesis has been suggested as a specific mechanism involved in cognitive (or neurogenic) reserve. The first objective of this study was to compare learning–related neuronal activity in adult-born versus developmentally born hippocampal neurons in juvenile male rats that had engaged in extensive running activity during early development or reared in a standard laboratory environment. The second objective was to investigate the long-term effect of exercise in rats on learning and memory of a contextual fear (CF) response later in adulthood. These aims address the important question as to whether exercise in early life is sufficient to build a reserve that protects against the process of cognitive aging. The results reveal a long-term effect of early running on adult-born dentate granule neurons and a special role for adult-born neurons in contextual memory, in a manner that is consistent with the neurogenic reserve hypothesis. The role of adult neurogenesis in learning and memory is under active investigation, but the underlying mechanisms remain unclear. The present study found that early-age running led to enhanced associative learning and memory in adult rats and increased activity of adult-born granule neurons in the dentate gyrus (DG) during memory retrieval. This study demonstrates the long-term effect of early-age physical activity on learning and memory much later in life. The findings emphasize the involvement of adult-born hippocampal neurons in neurogenic and functional cognitive reserve and show that physical activity contributes to memory improvement.Cognitive reserve refers to the brain’s capacity to draw on enriching experiences during youth to protect against adverse effects of structural decline, as in normal aging, and neuropathological damage resulting from accident or disease . A numbeThe relationship between cognitive protection and brain plasticity is central to the concept of cognitive reserve but our understanding of specific brain mechanisms involved is limited. One possibility may relate to adult neurogenesis, the capacity to produce new cells in the hippocampus that become integrated into neuronal networks of learning and memory. Several investigators have shown that physical exercise and other types of environmental enrichment increase neurogenesis levels  and imprIn previous studies, we used voluntary running to increase the levels of hippocampal neurogenesis in juvenile rats and examine neurogenesis levels at various time points, up to nine months later . VoluntaThe question addressed in the present study is whether voluntary running during early development can contribute to such a process in later adulthood. To test whether neurogenic reserve serves as a mechanism to improve memory, one group of rats was given voluntary access to running wheels for six weeks and another group was housed in standard laboratory cages. After four months rats were trained on a contextual fear (CF) conditioning task and, two weeks later, tested for memory of the CF response. CF is known to depend on the hippocampus and, in particular, new hippocampal neurons . Rats reTesting was done in the original conditioning context, a similar one, or a very different context, to provide information on the quality of memories. The purpose of manipulating context at test was to determine if these effects generalized to contexts that bore some similarity to the conditioning chamber but were not associated directly with shock. A novel aspect of the present study was the utilization of c-Fos-immunoreactivity to measure the activity of adult born neurons in the dentate gyrus (DG) in response to testing. Using immunohistochemical methods with a mitotic marker 5-chloro-2’-deoxyuridine (CldU) and an activity marker c-Fos, we were able to demonstrate the enhanced activity of adult-born dentate granule neurons in comparison to developmentally born neurons.n = 80) were housed for six weeks in specially designed cages with free access to running wheels, or in standard laboratory cages. After one week of acclimatization, the rats were divided into two groups, runners (n = 40) and non-runners (n = 40), and kept in single cages throughout the study. Runners and non-runners were maintained under the same housing conditions, including ad libitum access to food and water, except that cages of runners were fitted with a running wheel (circumference: 1.07 m). Running was monitored daily by means of a Vital View data acquisition system (Mini-Mitter A Respironics Company).One-month-old (juvenile) male rats . The running and non-running groups were subdivided into four subgroups. (1) No training and no test (controls). (2) Context A. (3) Context A’. And (4) context B. Three weeks before CF conditioning, all rats received a CldU injection. When the rats were six to seven months old, they were trained on a standard form of CF conditioning . Two weeAnimals were killed shortly after testing, and adult neurogenesis in the DG of the hippocampus was analyzed. Training and testing environments, as well as tissue collection, processing, and immunohistochemistry, are described in detail below. The experimental timeline is shown in Long Evans rats (Charles River) were used in this study. All animals were maintained on a 12/12 h light/dark cycle with lights on at 7 A.M. to 7 P.M. Animal weights were recorded regularly and animal procedures were in accordance with the guidelines of the Canadian Council on Animal Care. The experimental protocol was approved by the Animal Care Committee at the University of Toronto and Trent University.Running distances were monitored daily as well as the time of day that each animal spent running. The records showed that the rats predominantly ran during the dark phase of their cycle. Cumulative running distance or average distance traveled did not differ between cohorts designated to testing and control groups but the distance covered across the entire running period of six weeks increased progressively for all groups. Thus, rats ran an average of 21.8 (SD = 14.8) km during week 1 and reached the maximum of 71.1 (SD = 57.9) km during weeks five and six.All rats received CF conditioning in a wooden chamber (50 × 40 × 18 cm) that had four walls made of clear Plexiglas, a hinged clear Plexiglas roof with holes to allow ventilation, and a floor that consisted of metal rods, spaced 1.3 cm apart. The chamber was placed on a table, 1.3 m above the floor, and situated in the center of a standard laboratory room. The room contained standard furniture , as well as pictures, light fixtures, etc. on the walls. Illumination was provided by overhead fluorescent lights under rheostatic control.Training procedures for CF conditioning were similar to those followed in previous studies ; 2007. EFor testing, rats assigned to context A were tested in the same chamber and environment as in CF conditioning. Rats in context A’ were tested in the same chamber but the environment was slightly changed . Rats in the context B were tested in a smaller box (40 × 30 × 18 cm), also made of Plexiglas but with walls that were lined with opaque gray material. The roof was clear Plexiglas with ventilation holes and the floor consisted of metal rods, spaced 1.3 cm apart. This test box was placed in a different room on a table that was situated against a wall. Care was taken to ensure that the configuration of furniture, pictures, etc., was different from that of the room in which fear conditioning took place. Testing procedures were identical in all conditions. Testing consisted of a single trial in which the rat was placed in the appropriate box for 8 min, and in the absence of the tone, the amount of time spent freezing was recorded. The rat was then removed from the box and returned to its home cage.Three weeks before contextual fear conditioning (CFC) training, each rat was injected intraperitoneally with thymidine analog CldU . The injected solution was prepared by dissolving CldU in saline at 10 mg/ml and adjusting the pH to 7 with 0.5 µl of 10 N NaOH/ml saline. The injected dose was 85 mg of CldU/kg.Ninety minutes after testing animals were deeply anesthetized with isoflurane inhalation followed by transcardial perfusion with 300 ml ice-cold PBS followed by 300 ml ice-cold 4% paraformaldehyde (PFA). Following decapitation, brains were removed and placed in PFA for 24 h at 4°C. After postfix, brains were placed in PBS containing 0.1% sodium azide and stored until further processing.Brains were cut in half, and the hippocampus was dissected from the right hemisphere in each animal. Isolated hippocampi were sectioned along the dorso-ventral axis using a vibratome  into sections 30 μm thick. The sections were stored in PBS containing 0.1% sodium azide at 4°C until staining. Twelve sections were selected from each animal using a systematic random sampling procedure previously described . All imm2O), and coverslipped using PermaFluor (Thermo Scientific). Immunohistochemical controls included the omission of primary antibodies, which resulted in lack of staining at the corresponding wavelength in each instance.To identify cell survival and activity of newly formed cells, double-label immunohistochemistry was conducted for CldU and immediate early gene (IEG) protein c-Fos. Sections were incubated sequentially, with primary anti-c-Fos antibody , followed by secondary antibody donkey anti-rabbit IgG Alexa Fluor 568 , Life Technologies, A10042) in dark; afterward, to detect CldU, the tissue was incubated with primary antibody rat anti-BrdU , followed by secondary antibody goat anti-rat IgG Alexa Fluor 488 . This particular antibody can bind to CldU with high affinity . In all n = 80) CldU+ cells per animal were examined for double-labeling with c-Fos.All the single immunolabeled cells in the granule cell layer (GCL) zone of the DG were counted using a fluorescent microscope . Double-labeled neurons were visualized using Leica TCS SP5 confocal microscope (Leica Microsystems). The average number of cells per sampling section was multiplied by the number of hippocampal sections to yield the total number of cells per DG in each rat. Twelve sections were initially sampled from each animal. The sampling was repeated twice or in some animals three times to reach a minimum of at least 30 CldU+ cells per animal. On average 68.3 . The level of statistical significance was set at p ≤ 0.05.For immunohistochemistry and behavioral data, differences between groups were analyzed using two-way ANOVA with running and testing as variables. All pairwise multiple comparisons were conducted using the Holm-Sidak A necessary element of this study is a measure of overall activity in the hippocampal neurons, especially those in the DG. For this purpose, we used a c-Fos protein detected by immunohistochemistry as a measure of c-Fos gene expression. The results illustrated in 10.1523/ENEURO.0237-17.2017.1Figure 2-1f testing  = 10.838, *p = 0.001). There was no effect of running  = 1.131, p = 0.291) and no interaction  = 2.151, p = 0.101). Download Figure 2-1, TIF file.Activity-dependent regulation of c-Fos in the CA1 area of the hippocampus. Number (mean ± SEM) of c-Fos-labeled cells per CA1. Tested rats had significantly more cFos+ cells per CA1 compared to controls. Two-way ANOVA shows an effect oF = 15.9, p < 0.001) that was equal in runners and non-runners  = 0.297, p = 0.588) with no interactions among the variables  = 0.063, p = 0.979; A two-way ANOVA comparing c-Fos activation within the DG in early runners versus non-runners shows a testing-induced increase in c-Fos (p = 0.116) and testing (p = 0.883) as variables; data not shown). Thus, the test-induced increase in the hippocampal c-Fos activity is a true measure of the increased activity among the pre-existing neurons and not an effect on hippocampal growth.Additional estimates of granule cell volumes revealed no significant differences among the groups  were measured. The animals were injected with CldU three weeks before training thus labeling a cohort of five-week-old neurons at the time of testing. This age was chosen to ensure a full maturity and functionality of the neurons in terms of IEG expression .F = 5.837, p = 0.018), testing  = 13.946, p = 0.001) and  = 10.115, p = 0.002) interactions; The estimates demonstrate that the proportion of active cells is ∼3% in cage controls, and up to 7% in tested runners B. In terf testing. Context A representing the environment in which the rats were trained, A’ representing a similar, and B a completely novel environment  administered to animals at the time oironment .10.1523/ENEURO.0237-17.2017.2Figure 4-1F = 1.749, p = 0.19), testing  = 0.56, p = 0.453) and no interactions. Download Figure 4-1, TIF file.Cell survival in the DG. Graph showing number (mean ± SEM) of CldU-labeled cells per DG. No difference in the number of CldU+ cells was detected between groups. Two-way ANOVA shows no effect of running  = 23.57, p < 0.001), testing  = 5.582, p = 0.002), and significant interaction  = 2.872, p = 0.042; p < 0.05). Also, the cell numbers in tested runners were higher in comparison to those in tested non-runners. There were no differences among any of the non-running groups. Thus, the early-age running specifically enhances the activity of adult-born five-week-old neurons when rats are tested in the same, familiar or novel environment. Importantly, the percentage c-Fos expression is elevated in parallel to the absolute cells numbers. The increase shown in Two-way ANOVA performed on the absolute numbers of the double-labeled cells shows significant effects of running  = 11.672, p < 0.001) and testing  = 3.692, p = 0.031). The interactions between running and testing could not be adequately examined due to a low statistical power (0.05); however, a pairwise comparison with the Bonferroni t test revealed a significant difference between runners and non-runners within context A’ (p = 0.01) and B (p = 0.049) but not within A (p = 0.219).The animals were administered CF conditioning in context A two weeks before perfusion . Two-wayt = 1.027, 58 df, p = 0.31). In summary, these behavioral results show no effect of running on memory of the familiar context (A) but reduced freezing in the similar (A’) and completely different (B) contexts. The results parallel the c-Fos activation in adult-born granule DG neurons showing selective activation in runners but not in non-runners.These effects were specific for memory testing since the conditioning (training) phase showed no difference in learning. The percentage freezing scores in runners  and non-runners  were not different , is associated with hippocampal function  and, in Runners tested in context A also exhibited more activity in adult-born cells than non-runners, but the groups did not differ in freezing time. This outcome was not necessarily expected but is understandable when considering the dynamic changes that occur in CF memory over time . SeveralThe influence of early life experiences on learning and memory in later life is a topic needing more study . NumerouThere is considerable evidence that early physical and cognitive activity can delay the onset of pathologic changes that accelerate the process of cognitive decline in old age . In anim


Beta-sitosterol (BS) is a phytosterol, widely distributed throughout the plant kingdom and known to be involved in the stabilization of cell membranes. To compile the sources, physical and chemical properties, spectral and chromatographic analytical methods, synthesis, systemic effects, pharmacokinetics, therapeutic potentials, toxicity, drug delivery and finally, to suggest future research with BS, classical as well as on-line literature were studied. Classical literature includes classical books on ethnomedicine and phytochemistry, and the electronic search included Pubmed, SciFinder, Scopus, the Web of Science, Google Scholar, and others. BS could be obtained from different plants, but the total biosynthetic pathway, as well as its exact physiological and structural function in plants, have not been fully understood. Different pharmacological effects have been studied, but most of the mechanisms of action have not been studied in detail. Clinical trials with BS have shown beneficial effects in different diseases, but long-term study results are not available. These have contributed to its current status as an “orphan phytosterol”. Therefore, extensive research regarding its effect at cellular and molecular level in humans as well as addressing the claims made by commercial manufacturers such as the cholesterol lowering ability, immunological activity etc. are highly recommended. Beta-sitosterol (BS) is one of the several phytosterols with a chemical structure similar to that of cholesterol . It is aThe Joint FAO/WHO Expert Committee on Food Additives (JECFA) published scientific opinions on phytosterols without specific directives on BS separately . In a seThere are some reviews regarding the effects of phytosterols on health ,10,11 anBS has been reported to be present in various dietary and non-dietary plants . It exis29H50O, melting point is 139–142 °C and PubChem CID is 222284 . It is thermally unstable and converted to oxidized products [BS  is a whi29H48O , indicating a >C=C< system in the ring. A one proton broad multiplet at δH 4.44 showed a cross peak with C-2 protons and a C-4 proton in HETCOR and this signal was assigned to a C-3 methine proton. A plethora of multiplets was found in the range δH 1.1–2.14 which was informative in the presence of different methylene and methine protons of the steroidal structure. The other proton resonances were allocated to the glucopyranoside. Further evidence was also provided by the 13C NMR (detail is provided in ref. [The  in ref. ) that sh in ref.  carbon aO-β-d-glucopyranoside (β sitosterol glucoside) [The C3 carbon resonated at 71.73 ppm. The anomeric and the oxygenated methylene carbons of the sugar appeared at 100 and 61 ppm respectively. Thus on the basis of the spectral data, the structure of the compound was elucidated as stigmast-5-en-3-ucoside) .1H NMR spectrum  of BS (J = 6.4) at δ 5.36 that integrates for one proton. The spectrum further reveals two singlets at δ 0.68 and 1.01 ppm each of three proton intensity, assigned to two tertiary methyl groups at C-18 and C-19, respectively. The three proton intensity, is assigned to two tertiary methyl groups at C-18 and C-19, respectively. The NMR spectrum also displays two doublets (J = 6.4) at δ 0.83 and 0.81 which is attributable to two methyl groups at C-26 and C-27. The doublets (J = 6.5) at δ 0.93 are ascribed to a methyl group at C-21. On the other hand, the triplet at δ 0.84 (J = 7.2) of three proton intensity is assigned to the primary methyl group attached to C-29 [Another study showed that the 3) of BS  shows a  to C-29 .13C NMR together with COSY, HMQC, and HMBC shows twenty-nine carbon signals including six methyls, eleven methylenes, ten methane, and three quaternary carbons [The  carbons .Rf value of 0.55 when the crystals were reconstituted in chloroform and spotted on the TLC plate in n-hexane: acetone (80:20) mobile phase system [In Thin layer chromatography (TLC) analysis of BS revealed a e system .To analyze BS, a Gas-liquid chromatography (GLC) method using the butyl ester of BS was implemented . It was Different High-Performance Liquid Chromatography (HPLC) methods have been developed for analyzing BS such as a narrow-bore HPLC-UV method ; a high-Ahmida et al. (2006) developed capillary gas chromatography coupled to mass spectrometry (GC-MS) for simultaneous detection of BS from other sterols by multiple selected ion monitoring. This method is based on the alkaline hydrolysis of sterol esters, extraction of free sterols and derivatization. The recovery of all sterols was in the range 76%–101% . Most of13C-labeling approach, the mechanism of BS biosynthesis was studied and it was proposed that isopentenyl diphosphate (IPP) combines with dimethylallyl diphosphate (DMAPP) to form farnesyl diphosphate (FPP) and then two molecules of FPP combine tail-to-tail to form Squalene, a triterpene and then cycloartenol which eventually forms BS by methylation, hydride shift, reduction, and slight modification in the beta-ring [The exact biosynthetic mechanism of BS varies according to organisms, but generally, it follows the mevalonate pathway . BS is beta-ring .S-Adenosyl methionine (SAM) to give a carbocation that undergoes a hydride shift and loses a proton to yield an intermediate compound with a methylene side-chain. Both of these steps are catalyzed by sterol C-24 methyltransferase. The intermediate compound is then catalyzed by sterol C-4 demethylase and loses a methyl group to produce cyclo-eucalenol. Subsequent to this, the cyclopropane ring is opened with cyclo-eucalenol cyclo-isomerase to form another intermediate compound. This intermediate compound then loses a methyl group and undergoes an allylic isomerization to form gramisterol. This step is catalyzed by sterol C-14 demethylase, sterol Δ14-reductase, and sterol Δ8-Δ7-isomerase. The last methyl group is removed by sterol demethylase to form episterol. Finally, episterol is converted to β-sitosterol through methylation by SAM, reduction by NADPH, and modifications in the β-ring. Here 24-methylenesterol C-methyltransferase plays a very important role [Both the biosynthesis of BS and Cholesterol follow the same direction till the formation of Squalene . Howeverant role   Synthesis of isopentenyl pyrophosphate, the “building block” of cholesterol; (2) Formation of squalene via the condensation of six molecules of isopentenyl phosphate and; (3) Conversion of squalene to cholesterol via several enzymatic reactions  Figure .20−21 alkene was found to produce BS contaminated with varying amounts of recovered stigmasterol as well as the fully saturated stigmastanol [5−6 alkene as cyclopropyl carbinyl ether. Following hydrogenation of the Δ22−23 double bond, solvolysis of the cyclopropane reintroduces both the C3-alcohol and the Δ5−6 alkene [5−6 alkene as an epoxide [Different routes have been reported for the synthesis of BS over more than 50 years. The selective hydrogenation of the stigmasterol side chain Δmastanol ,44. In a6 alkene ,46. Rece epoxide . HoweverBS containing plants show antinociceptive , anxiolyAccording to the Norwegian Food Safety Authority , BS has a skin conditioning effect and is used in sunscreen, moisturizer, body wash, and anti-aging cosmetic preparations . Skin is one of the paths of BS excretion. It has been reported that BS inhibits the production and mRNA expression of thymic stromal lymphopoietin through blocking of caspase-1 and nuclear factor-kB (NkB) signal pathways in the stimulated human mast cell line, HMC-1 cells. Even though this study showed the potential therapeutic effect against atopic dermatitis, studies on long-term use of BS on the skin need to be conducted .BS has beneficial effects on the cardiovascular system and prevents different cardiovascular diseases except for patients with ABCG5 and ABCG8 mutation ,58. HoweBS containing diets change the live ultra-structure and such differences are observed in both young and adult mice fed with BS . PathophBS possesses a weakly estrogenic effect and alone or in combination with progesterone, it inhibits the expression of intercellular adhesion molecule-1  and testThe effect of BS on the reproductive system is contradictory. Study on American mink shows increased male fertility due to BS intake , but othMimosa tenuiflora have been used for decades as a remedy in the treatment of wounds and burns of the skin. This can be explained by the re-epithelialization process in wounded areas which is believed to be aided by BS. So, the ability to heal, together with the anti-inflammatory and antimicrobial activity of BS demonstrate its potential in tissue engineering applications [Different plants containing phytosterols like ications .Pharmacokinetics and the bioavailability of BS  have beeStructurally cholesterol and BS are different from each other only by the additional ethyl group at the C-24 position in the latter. The absorption of BS is one-fifth of that of cholesterol . Field aSeveral findings suggest that BS has antioxidant property ,101. It BS plays a role in blood vessel formation and thus possesses potentials in wound healing . HoweverBS is recommended for the prevention of different cardiovascular diseases ,107,108 A study on rats has shown that the antipyretic effect of BS is comparable to that of aspirin . The prel-type voltage-dependent calcium channels, phosphoinositide kinase-3, intracellular calcium and microtubule modulation, and thus promotes the anti-inflammatory activity as proposed by Liz et al. (2013) [BS possesses anti-inflammatory activity in human aortic cells  as well . (2013) , require. (2013) .It has been reported from a study that the plant extract containing BS has significant anti-arthritic activity . AccordiBS has been shown to act as a powerful immune modulator . BS exhiExperimental and epidemiological studies have shown the efficacy of BS in treating different types of cancer via different pathways. One recent review documented in detail regarding this . HoweverOral treatment with BS increases the fasting plasma insulin levels. There is a corresponding decrease in fasting glycemia when BS is administered orally. In addition, it improves the oral glucose tolerance test with an increase in glucose-induced insulin secretion . These eAccording to the double-blind, randomized, placebo-controlled trial conducted by Donald et al. (1997) on culture-positive pulmonary tuberculosis patients, BS has been found to have a significant improvement in weight that is lost due to pulmonary tuberculosis . The samO-glucopyranoside (BSG), a derivative of BS, inhibits bacterial cell adhesion to a fibronectin indicating that modification of BS is needed to exert its antibacterial effect [BS obtained from different plants shows antibacterial and antifungal activity without toxicity in brine shrimp lethality assay ,170,171.l effect . HoweverBS has been reported to have anthelmintic properties alone  and in c50 in rats (>2 gm/kg) [Even though the United States National Toxicology Program (NTP) reviewed toxicological effects of BS about 18 years ago , many study results need to be re-evaluated based on the latest findings. A high level of BS concentrations in blood has also been correlated with increased severity of heart disease in men who have previously been suffering from heart attacks . There a2 gm/kg) , prevent2 gm/kg) , and sho2 gm/kg) . Even th2 gm/kg) , later s2 gm/kg) . However2 gm/kg) . AnotherSide chain double bonds increased sterol mobility considerably in HPLC, which reflected decreased hydrophobicity of the molecule. However, the change in hydrophobicity depended upon the position of the side chain double bond: sterols with double bonds at the C22 position were more hydrophobic than sterols with double bonds at the C24 position. Increases in the side chain length, by the addition of methyl or ethyl groups, resulted in decreased HPLC mobility and increased hydrophobicity, whereas the insertion of a double bond in one or both fatty acyl chains decreased hydrophobicity . BS has BS has been reported to have beneficial effects in different diseases, but it has not developed as an independent drug mostly because of its relatively lower efficacy and the development of other drugs with higher efficacy. For example, both BS and glucocorticoid, dexamethasone (DX) have anti-inflammatory effects, but DX has obtained unprecedented approval being a standard drug since its inception  even tho


Pseudomonas chlororaphis HT66 isolated from the rice rhizosphere is an important plant growth-promoting rhizobacteria that produce phenazine-1-carboxamide (PCN) in high yield. Phenazine production is regulated by a quorum sensing (QS) system that involves the N-acylated homoserine lactones (AHLs)—a prevalent type of QS molecule.N-(3-hydroxy hexanoyl)-l-homoserine lactone (3-OH-C6-HSL), N-(3-hydroxy octanoyl)-l-homoserine lactone (3-OH-C8-HSL) and N-(3-hydroxy decanoyl)-l-homoserine lactone (3-OH-C10-HSL). The signal types and methods of synthesis were different from that in other phenazine-producing Pseudomonas strains. By non-scar deletion and heterologous expression techniques, the biosynthesis of the AHL-signals was confirmed to be only catalyzed by PhzI, while other AHLs synthases i.e., CsaI and HdtS were not involved in strain HT66. In comparison to wild-type HT66, PCN production was 2.3-folds improved by over-expression of phzI, however, phzI or phzR mutant did not produce PCN. The cell growth of HT66∆phzI mutant was significantly decreased, and the biofilm formation in phzI or phzR inactivated strains of HT66 decreased to various extents.Three QS signals were detected by thin layer chromatography (TLC) and high-performance liquid chromatography–mass spectrometry (HPLC–MS/MS), which identified to be PhzI–PhzR system plays a critical role in numerous biological processes including phenazine production.In conclusion, the results demonstrate that The online version of this article (10.1186/s12934-017-0854-y) contains supplementary material, which is available to authorized users. N-acylated homoserine lactones (AHLs) to sense cell density, which is composed of a homoserine lactone ring (HSL) with varying acyl chain + (266.1363) were also observed and confirmed that the AHL was 3-OH-C8-HSL. Combined with the results of TLC and bio-reporter experiments, we concluded that the AHLs produced by HT66 were 3-OH-C6-HSL, 3-OH-C8-HSL, and 3-OH-C10-HSL.Further, ethyl acetate extracts from HT66 culture supernatants were separated by C18-reverse TLC and then overlaid with the biosensor NTL4 strain. Notably, three blue spots were detected from the extracts of HT66  to the AHL synthesis gene of PCL1391. This gene encodes 196 amino acids, of which only one amino acid is different from phzI of PCL1391. At the same time, an HdtS homolog was discovered, which has a similarity of 83% with the HdtS in P. fluorescens F113. The hdtS encodes 257 amino acids that direct the synthesis of C6-HSL, C10-HSL, and 3OH-C14:1-HSL in F113. Additionally, we also found another possible AHL synthesis gene in HT66 genome, CsaI, which had been reported to produce C4-, C5- and C6-HSL in P. chlororaphis 30-84 [CsaI gene only contains 204 bp but shows high identity (over 93%) to the csaI (1416 bp) in strain 30-84. The CsaI–CsaR is a second quorum sensing system in 30-84, which is supposed to regulate the cell surface properties in response to the signals produced by CsaI.The whole-genome shotgun sequence of the HT66 has been deposited in National Center for Biotechnology Information (NCBI) databases under accession number ATBG00000000. In order to study which gene participated in quorum sensing and phenazine biosynthesis, is 30-84 . AlthougphzI, casI and hdtS were cloned and heterologous expressed in E. coli DH5α. A broad-host-range expression vector pME 6032 was used and the target gene was inserted between the EcoRI and XhoI sites, which was located in the downstream of the Ptac promoter. After induction with IPTG, the DH5α with plasmid pME-phzI, pME-csaI or pME-hdtS were cultivated overnight; the culture broths were then extracted with ethyl acetate and detected by A. tumefaciens NTL4 plate assay. Only the pME-phzI culture sample contained a detectable amount of AHLs  in HT66, which are irrelevant with CsaI or HdtS.To confirm our conclusion, we also constructed ∆phzI, and phzR are located just upstream of the phenazine biosynthesis gene cluster. In this work, we knocked out phzI from the HT66 genome to construct HT66∆phzI mutant through a non-scar deletion method. The results showed that deletion of phzI caused the complete loss of PCN biosynthesis as well as the AHLs synthesis . Thus, the results implied that exogenous AHLs production had improved the PCN production.To investigate the effects of exogenous AHLs on PCN production in HT66, we transformed the phzI or ∆phzR mutants indicated that a functional expressed quorum-sensing system was essential for the activation of the phz operon. The continuous over-expression of phzI resulted in an elevated level of PCN in wild-type HT66 strain. In addition, the PCN titers of ∆csaI and ∆hdtS mutants were detected to be 425 and 469 mg/l, respectively, and had statistically no difference in contrast with the wild-type HT66 strain.The absence of PCN production in ∆phzR mutant. While ∆phzI mutant had a lower cell density in stationary phase compared to wild-type, which might be correlated with the absence of signals and negative impact on communication between microorganisms.A comparative growth profile results portrayed that the HT66 strain reached stationary phase at almost 24 h, and started to decline after 48 h Fig. a. No notP. aeruginosa biofilm development. On the other hand, QS signaling and transcription of genes are connected with the biofilm matrix biosynthesis [phzI and ∆phzR mutants , the mutants had almost the identical growth rate as the wild-type to form colonies  and has a relationship with the lysophosphatidic acid acyltransferase family [In this study, thin-layer chromatography and liquid chromatography-mass/mass spectrometry techniques were applied to identify three AHLs  in ynthesis . In P. fe family .phzI in several Pseudomonas were compared with that in HT66, including their signals products. PhzI proteins of HT66 and PCL1391 were found to be similar as high as 99% . Noticeably, phenazine production was supposed to be strongly stimulated by PhzI-mediated AHLs than produced by AurI and CsaI that only induced a slight stimulation. Though, Pseudomonas sp. CMR12a and P. chlororaphis subsp. aureofaciens 30-84 display a second QS system apart from phzI/phzR system, phenazine biosynthesis was merely regulated by the AHLs produced by PhzI [Apart from the identification and production of AHLs in HT66, this study found that the over-expression of i et al.  reported by PhzI , 42, 43.P. ultimum was used in this study. After cultivation together for 2 weeks, the development of pathogenic fungi was considerably inhibited by the wild-type presumably due to PCN production confirming the strain as an effective biocontrol agent. In P. chlororaphis PA23, it was examined that the biofilm development was reduced by approximately fivefold in the QS-deficient mutants and the motility was also altered [phzI and phzR deletion strains of HT66 had a decrease in different extents. With the absence of PCN, the antimicrobial ability was influenced when grew against the P. ultimum in vitro.In order to appraise the involvement of AHL production to the biocontrol capacity of StFRB508,  altered . SimilarP. chlororaphis HT66 by TLC and HPLC–MS/MS analyses. The production of AHLs exhibiting the activating role in phenazine biosynthesis gene cluster was only catalyzed by PhzI. In comparison to wild-type HT66, PCN production was 2.3-folds improved by over-expression of phzI, however, phzI or phzR mutant did not produce PCN. In addition, the growth, morphology and biofilm formation were all under the control of the PhzI–PhzR regulatory system. These results revealed that PhzI–PhzR system plays a pivotal role in PCN production and the biocontrol activity of HT66.In conclusion, we identified three kinds of AHLs  in Additional file 1: Figure S1.a Colonial morphology changes in wild-type HT66, HT66∆phzI and HT66∆phzR strains during 7 days and b Influence of antifungal activity of HT66 and its mutants on the growth of Pythium ultimum. P. ultimum was spotted at the left of the PDA plate, whereas HT66 and its mutants were inoculated on the right side.